Translocation Of Notch Intracellular Domain Research Articles

Didymin ameliorates ulcerative colitis-associated secondary liver damage by facilitating Notch1 degradation

BackgroundDidymin is a dietary flavonoid originally discovered by our group as a potent anti-ulcerative colitis (UC) agent. However, whether didymin plays a protective role in UC-associated inflammatory liver injury is still unclear. PurposeThis study aimed to evaluate the therapeutic potential of didymin on UC-associated inflammatory liver injury and explore the underlying mechanism. Study design and methodsColitis model was established in C57BL/6 mice by exposure to DSS, and didymin was administrated intragastrically for consecutive 10 days. The inflammatory liver injury was assessed by levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum and histopathological damage in the liver. In vitro Kupffer cells and RAW264.7 cells challenged with lipopolysaccharides (LPS) were used to explore the modulatory activity of didymin on pro-inflammatory cytokines secretion and Notch1 signaling pathway activation. ResultsDidymin significantly mitigated liver coefficiency, ALT and AST levels in serum, and the hepatic histopathological damage caused by DSS-induced acute and chronic colitis. The mRNA expressions of pro-inflammatory factors including Tnf, Il1, and Il6 in liver tissues, Kupffer cells, and RAW264.7 cells stimulated by the influx of LPS was significantly deprived after didymin treatment. Mechanistically, didymin obstructed the protein expression, nuclear translocation of notch intracellular domain 1 (Notch1-ICD) and mRNA expression of hairy and enhancer of split 1 (Hes1). Further, the inhibitory mechanism of the Notch1-Hes1 pathway was dependent on c-Cbl-mediated Notch1-ICD lysosomal degradation. ConclusionOur study verified for the first time that didymin could prevent UC-associated diseases, such as inflammatory liver injury, and the mechanism was related to facilitating Notch1 lysosomal degradation rather than proteasome degradation via promoting protein expression of c-Cbl in macrophages. Our findings that the inhibition of Notch1 signaling transduction helps to alleviate UC-associated liver injury provides possible therapeutics for the treatment of colitis and also furnishes a research paradigm for the study of flavonoids with similar structures.

DLX5 regulates the osteogenic differentiation of spinal ligaments cells derived from ossification of the posterior longitudinal ligament patients via NOTCH signaling.

Ossification of the posterior longitudinal ligaments (OPLL) is common disorder characterized by heterotopic ossification of the spinal ligaments. Mechanical stimulation (MS) plays an important role in OPLL. DLX5 is an essential transcription factor required for osteoblast differentiation. However, the role of DLX5 during in OPLL is unclear. This study aims to investigate whether DLX5 is associated with OPLL progression under MS. Stretch stimulation was applied to spinal ligaments cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. Expression of DLX5 and osteogenesis-related genes were determined by quantitative real-time polymerase chain reaction and Western blot. The osteogenic differentiation ability of the cells was measured using alkaline phosphatase (ALP) staining and alizarin red staining. The protein expression of DLX5 in the tissues and the nuclear translocation of NOTCH intracellular domain (NICD) was examined by immunofluorescence. Compared with non-OPLL cells, OPLL cells expressed higher levels of DLX5 in vitro and vivo (p < 0.01). Upregulated expression of DLX5 and osteogenesis-related genes (OSX, RUNX2, and OCN) were observed in OPLL cells induced with stretch stimulation and osteogenic medium, whereas there was no change in the non-OPLL cells (p < 0.01). Cytoplasmic NICD protein translocated from the cytoplasm to the nucleus inducing DLX5 under stretch stimulation, which was reduced by the NOTCH signaling inhibitors (DAPT) (p < 0.01). These data suggest that DLX5 play a critical role in MS-induced progression of OPLL through NOTCH signaling, which provides a new insight into the pathogenesis of OPLL.

A novel triazole, NMK-T-057, induces autophagic cell death in breast cancer cells by inhibiting γ-secretase–mediated activation of Notch signaling

Notch signaling is reported to be deregulated in several malignancies, including breast, and the enzyme γ-secretase plays an important role in the activation and nuclear translocation of Notch intracellular domain (NICD). Hence, pharmacological inhibition of γ-secretase might lead to the subsequent inhibition of Notch signaling in cancer cells. In search of novel γ-secretase inhibitors (GSIs), we screened a series of triazole-based compounds for their potential to bind γ-secretase and observed that 3-(3'4',5'-trimethoxyphenyl)-5-(N-methyl-3'-indolyl)-1,2,4-triazole compound (also known as NMK-T-057) can bind to γ-secretase complex. Very interestingly, NMK-T-057 was found to inhibit proliferation, colony-forming ability, and motility in various breast cancer (BC) cells such as MDA-MB-231, MDA-MB-468, 4T1 (triple-negative cells), and MCF-7 (estrogen receptor (ER)/progesterone receptor (PR)-positive cell line) with negligible cytotoxicity against noncancerous cells (MCF-10A and peripheral blood mononuclear cells). Furthermore, significant induction of apoptosis and inhibition of epithelial-to-mesenchymal transition (EMT) and stemness were also observed in NMK-T-057-treated BC cells. The in silico study revealing the affinity of NMK-T-057 toward γ-secretase was further validated by a fluorescence-based γ-secretase activity assay, which confirmed inhibition of γ-secretase activity in NMK-T-057-treated BC cells. Interestingly, it was observed that NMK-T-057 induced significant autophagic responses in BC cells, which led to apoptosis. Moreover, NMK-T-057 was found to inhibit tumor progression in a 4T1-BALB/c mouse model. Hence, it may be concluded that NMK-T-057 could be a potential drug candidate against BC that can trigger autophagy-mediated cell death by inhibiting γ-secretase-mediated activation of Notch signaling.

Inhibition of nuclear translocation of notch intracellular domain (NICD) by diosgenin prevented atherosclerotic disease progression

Notch signaling plays a pivotal role in homeostasis and cardiovascular development. The role of notch signaling in atherosclerosis cannot be complete without analysing the key role of notch in macrophages, which trigger the inflammatory response and subsequent plaque formation in atherosclerosis. Diosgenin showed its anti-atherosclerotic property by the unifying mechanism of suppressing the expression of notch signaling pathway, particularly the nuclear translocation of notch intracellular domain (NICD) in aorta and in differentiated macrophage cells. It is further confirmed by the inhibition of NICD by DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), which also restricted the differentiation of macrophage. Hence, inhibition of nuclear translocation of NICD by diosgenin aids in preventing atherosclerosis.

Abstract P6-15-03: Phase Ib Trial of the Gamma Secretase Inhibitor (GSI), MK-0752 Followed by Docetaxel in Locally Advanced or Metastatic Breast Cancer

Abstract Background: The cancer stem cell hypothesis asserts that there is a small population of cells within a tumor that has the ability to self renew and differentiate, and that these cells drive tumor growth and metastasis but are resistant to conventional cytotoxic chemotherapy. Pathways involved in stem cell growth and differentiation are viable targets for new anticancer therapies. One such pathway, Notch, is inhibited by GSIs which prevent translocation of Notch intracellular domain to the nucleus. Inhibition of GS concurrent with chemotherapy might improve disease control by targeting both stem cells and differentiated cells within the tumor. This Phase Ib clinical trial was designed to determine the MTD of the GSI, MK-0752, in combination with docetaxel, and to evaluate an effect on stem cell markers in serial tumor biopsies. Methods: Eligible subjects had metastatic breast cancer or locally advanced breast cancer that did not respond to anthracycline therapy. Patients with disease that progressed on a taxane, or who had received a taxane within 6 months were excluded. MK-0752 was administered orally on days 1-3 of each 21-day cycle of therapy, in escalating doses. Dose levels (mg/day) 1=300; 2=450; 3=600; and 4=800. Docetaxel 80 mg/m2 IV was administered day 8, with pegfilgrastim day 9 each cycle. Treatment was continued until disease progression, unacceptable toxicity, or symptomatic deterioration. The trial was monitored using the Time to Event Continual Reassessment Method, targeting a 20% toxicity rate. Tumor biopsies were performed at baseline, after 1 cycle, and at treatment completion in a subset of patients. Results: 30 patients were enrolled between Mar 2008 and Jan 2010. Dose limiting toxicities of the combination included diarrhea, hand-foot syndrome, and LFT elevation. 20/30 patients experienced Grade 1 or 2 fatigue. The final estimates and confidence intervals for the probability of dose limiting toxicity at each dose level are summarized in the table: Probability = probability of dose-limiting toxicity 20 enrolled patients had measurable disease by RECIST criteria. Of these, 9 had PR, 8 SD, and 3 PD, for an estimated RR of 45% to the combination. 2 patients have been maintained on therapy in excess of 22 cycles. Conclusions: Dose level 3 was identified for further study in a Phase II randomized trial. Efficacy of docetaxel was not inhibited by MK-0752, as a 45% RR in patients with measurable disease was observed. There is intriguing long term disease stabilization in 2 patients. Evidence of an effect of the combination on the stem cell population was apparent on serial biopsies as presented at SABCS Dec 2009 (Abstract # 48); additional biopsy data will be presented. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-03.

Dynamic intracellular distribution of Notch during activation and asymmetric cell division revealed by functional fluorescent fusion proteins

Notch signaling mediates intercellular communication required for cell fate specification. Because the majority of Notch proteins exist as an intracellular pool, information on intracellular trafficking of Notch is essential for understanding its regulation. To observe trafficking of Notch protein in vivo, we constructed a Notch protein with green fluorescent protein (GFP) inserted into the intracellular domain. This construct rescued the neurogenic phenotype of Notch mutant embryos and caused the typical Notch gain-of-function phenotype when over-expressed in the wing imaginal disc. Using this and similarly constructed Notch-mCherry fusion, we showed rapid spatio-temporal dynamics of Notch in endosomes. Kinetic analysis revealed that activation of Notch-GFP by ectodomain shedding causes fast and unrestricted nuclear translocation of Notch intracellular domain. During asymmetric cell division of sensory organ precursor cells, distribution of Notch-GFP showed transient asymmetry at the interface of daughter cells, confirming a possible site of regulation that biases Notch signaling level among daughter cells.

NUMB is a break of WNT - Notch signaling cycle

Notch, FGF and WNT signaling pathways cross-talk during embryogenesis, tissue regeneration and carcinogenesis. Notch-ligand binding to Notch receptors leads to the cleavage of Notch receptors and the following nuclear translocation of Notch intracellular domain (NICD) to induce transcriptional activation of Notch target genes. Notch signaling inhibitors, NUMB and NUMB-like (NUMBL), are docking proteins with PTB domain. We searched for the TCF/LEF-binding site within the promoter region of NUMB and NUMBL genes. Because two TCF/LEF-binding sites were identified within human NUMB promoter based on bioinformatics and human intelligence (Humint), comparative integromics analyses on NUMB orthologs were further performed. Chimpanzee NUBM gene, consisting of 13 exons, was identified within NW_115880.1 genome sequence. XM_510045.1 was not the correct coding sequence for chimpanzee NUMB. Chimpanzee NUMB gene was found to encode a 651-amino-acid protein showing 99.5, 93.9 and 82.6% total-amino-acid identity with human NUMB, mouse Numb and chicken numb, respectively. Human NUMB mRNA was expressed in placenta, ES cells, neural tissues, trachea, testis, uterus, thymus, coronary artery as well as in a variety of tumors, such as cervical cancer, tong tumor, brain tumor, colorectal and breast cancer. Although distal TCF/LEF-binding site within human NUMB promoter was conserved only among primate NUMB orthologs, proximal TCF/LEF-binding site was conserved among primate and rodent NUMB orthologs. NUMB, JAG1, FGF18, FGF20 and SPRY4 are potent targets of the canonical WNT signaling pathway in progenitor cells. NUMB inhibits Notch signaling in progenitor cells to induce differentiation, while JAG1 activates Notch signaling in stem cells to maintain self-renewal potential. Because Notch signaling inhibitor NUMB was identified as the safe apparatus for the WNT - Notch signaling cycle, epigenetic silencing, deletion and loss-of-function mutation of NUMB gene could lead to carcinogenesis through the dysregulation of the WNT - Notch signaling cycle.

Notch ligand, JAG1, is evolutionarily conserved target of canonical WNT signaling pathway in progenitor cells

WNT, Notch, FGF, and Hedgehog signaling pathways network together during embryogenesis, tissue regeneration, and carcinogenesis. Association of Notch ligands with Notch receptors on neighboring cells leads to cleavage of Notch receptors by metalloprotease and gamma-secretase to induce nuclear translocation of Notch intracellular domain (NICD). Nuclear complex, consisting of CSL (RBPSUH), NICD, Mastermind (MAML), p300 and histone acetyltransferase (HAT), then induces transcriptional activation of Notch target genes, such as HES1, HES5, HES7, HEY1, HEY2 and HEYL. Here, we searched for TCF/LEF-binding site within the promoter region of Notch ligand genes, including DLL1, DLL3, DLL4, JAG1 and JAG2. Because TCF/LEF-binding sites were identified within human JAG1 promoter based on bioinformatics and human intelligence, comparative genomics analyses on JAG1 orthologs were further performed. Chimpanzee JAG1 gene, consisting of 26 exons, was identified within NW_120319.1 genome sequence. XM_525264.1 and XM_514517.1 were not the correct coding sequences for chimpanzee JAG1. Chimpanzee JAG1 gene was found to encode a 1218-amino-acid protein showing 99.5% and 96.2% total-amino-acid identity with human JAG1 and mouse Jag1, respectively. Phylogenetic analysis revealed that JAG1 orthologs were more conserved than those of other Notch ligands. JAG1 gene was identified as evolutionarily conserved target of WNT/beta-catenin signaling pathway based on the conservation of double TCF/LEF-binding sites within 5'-promoter region of mammalian JAG1 orthologs. Human JAG1 mRNA was expressed in embryonic stem (ES) cells, neural tissues, lung carcinoid, gastric cancer, pancreatic cancer, colon cancer, and also in squamous cell carcinoma (SCC) of skin, oral cavity, esophagus, head and neck. JAG1 expression on progenitor cells due to canonical WNT signaling activation induces self-renewal of stem cells due to Notch signaling activation. JAG1, functioning as WNT-dependent Notch signaling activator, is the key molecule maintaining the homeostasis of stem and progenitor cells.

Identification and characterization of human HESL, rat Hesl and rainbow trout hesl genes in silico.

Activation of Notch signaling pathway leads to nuclear translocation of Notch intracellular domain (NIC), and transcriptional activation of target genes through the interaction between CSL proteins (RBPSUH or RBPSUHL) and NIC. HES1, HES5, HEY1 and HEY2 are Notch target genes. Mammalian HES/HEY family proteins as well as Drosophila Hairy and Enhancer of split are implicated in the cell fate determination. We have previously identified and characterized human HES2, HES3 and HES5 genes. Here, we identified and characterized human HES-like (HESL), rat Fesl, and rainbow trout fesl genes by using bioinformatics. Human HESL gene, located within AC093824.3 genome sequence, was mapped to human chromosome 4q35.1 between ACSL1 and SLC25A4 genes. Rat Fesl gene, located within AC111303.4 genome sequence, was mapped to rat chromosome 16q11. EST BX875070.2 was the representative rainbow trout fesl cDNA. HESL-ACSL1 locus was conserved among human, rat, mouse, and zebrafish genomes. Human HESL (241 aa) showed 92.9% total-amino-acid identity with rat Hesl (240 aa), 92.1% total-amino-acid identity with mouse Hesl (240 aa), and 68.0% total-amino-acid identity with rainbow trout hesl (235 aa). HESL orthologs consist of basic Helix-loop-helix (bHLH) domain and ORANGE domain. C-terminal region of HESL orthologs were divergent from that of HES, HEY, and DEC homologs. Phylogenetic analyses revealed that bHLH transcription factors with ORANGE domain were classified into the following three groups: (group I) HES1, HES2, HES4 and HES6 orthologs; (group II) HESL, HES5, HES7, HEY1, HEY2 and HEYL orthologs; (group III) HES3, DEC1/BHLHB2 and DEC2/BHLHB3 orthologs.

Induction of Notch signaling by tumor necrosis factor in rheumatoid synovial fibroblasts

Rheumatoid arthritis (RA) is characterized by progressive inflammation associated with abberrant proliferation of synoviocytes. In order to explore the characteristics of rheumatoid synovial fibroblasts (RSF), we performed the comparative gene expression profile analysis between RSF and normal synovial fibroblasts (NSF) upon tumor necrosis factor (TNF) stimulation. As an initial screening for the genes preferentially induced by TNF in RSF compared with NSF, we have adopted a cDNA array containing well-defined sets of genes responsible for cell growth, cell fate determination, and cellular invasiveness. Differentially expressed genes of interest were confirmed using real-time RT-PCR. We found that TNF induced the expression of Notch-1, Notch-4, and Jagged-2 in RSF. The expression of these proteins was detected in the RA synovial tissues. The nucleus of RA synoviocytes showed strong staining with anti-Notch-1 and Notch-4 antibody. TNF induced the nuclear translocation of Notch intracellular domain in RSF, indicating the elicitation of the Notch signaling. Notch-1, Notch-4, and Jagged-2 proteins were also detected in the developing synovium of mouse embryo. Thus, RSF may have re-acquired the primordial phenotype, accounting for the hyperproliferation and aggressive invasiveness, exhibiting tumor-like phenotype.